To perform resequencing chip analysis in patients with isolated eye abnormalities collected in earlier studies: This project started several years ago in the OGFVB/NEI. We developed a screening tool by the resequencing CHIP on 93 genes provided an opportunity for these samples from patients enrolled in NEI clinical research for years. With Dr. Miller and Dr. Sohrabys help, we were able to continue the project using core facility in NNRL/NEI by Dr. Swaroop and Matthew Brooks. We eventually established the testing protocol and tested first group of samples provided by eyeGENE program. Our manuscript was published in the IVOS later in 2011. We analyzed more patients by a graduate student, Dr. Xi Huang who was sponsored by Chinese Government Fellowship. In the last year, we have analyzed 22 patients with isolated RP using this technology. We were collaborated with Dr. Fanns group from ITBP/NINDS in data processing and bioinfomatic analysis. With selected candidate variations, we were verifying candidate variations by Sanger sequencing in patient samples with reference DNA by Dr. Danyao Nie, a visiting ophthalmologist from China. We are summarizing the data and preparing for manuscript. To develop new screening tool for retinal disease related genes: Retinitis Pigmentosa (RP) is genetically heterogeneous. RP patients have been found with mutations in more than 54 genes. Still more genes were and will be identified as responsible for RP from time to time. Molecular technology has been moved into next generation sequencing era. To develop a more advanced study tool, we have explored new approaches. We have selected a new technology called RainStromTM Microdroplet PCR to replace regular high-throughput PCRs in our resequencing chip protocol for target genomic DNA enrichment. We hired a company, RainDance Technologies, Inc., in designing a primer library called Retinal Dystrophy Panel (RD panel) which covers the genomic DNA regions of coding exons in 184 genes related to retinal function and development. Using the newly developed primer library, RainDance Technologies, Inc also performed target DNA regions enrichment in 16 samples including 13 RP patients, 2 CRD patients and one CEPH reference DNA. With helps from core facility in NNRL/NEI by Dr. Swaroop and Matthew Brooks, we have sequenced the enriched samples using GAIIx, a next generation sequencer. Again, Dr. Fanns group from ITBP/NINDS helped the data processing and bioinfomatic analysis. We have identified possible candidate mutations from identified variations. In this summer, a summer intern, Ms. Candice Craig, has been working on verification of the identified targets by Sanger sequencing. We are summarizing the data and preparing for manuscript. We are continuing the procedures to analyze more patients with RP. Another cohort of 24 samples has been processed for PCR enrichment using RDT1000 in the Dr. Eric Hoffmans lab in the Childrens National Medical Center. Those samples have been sequenced in our newly purchased MiSeq next generation Sequencer from Illumina. The data is now in processing and bioinformatic analysis at ITBP/NINDS. Thanks to NEI OSD Dr. Miller and Dr. Sohrabys supports, and NEI users committee, we have been approved to invest the advanced RainStrom techonology, Thunderstorm system and the next generation sequencer. With the MiSeq and Thunderstorm system, we will be able to perform the long time waited project: High-throughput Mutation Screening in Inherited Retinal Dystrophies. We are planning to screen 100 samples from patients with retinal disorders enrolled in the eyeGENE program. Our plan is to validate the procedure to a clinical test in next year upon successfully finishing this project. To analyze the PAX2 gene structure in patients collected in earlier studies: This is a continuation of my clinical ophthalmic research from Minnesota and collaboration. We developed a clinical DNA testing of PAX2 gene for Renal-coloboma Syndrome and have been collecting tested samples for genetic heterogeneity analysis. After I joined NEI, we are extending this study. We have collaborated with Dr. Brain Brooks to study 7 patients in their PAX2 genes. We are extending the study with a hope that we could validate this protocol to a clinical test. To identify the breakpoints of RS1 gene partial deletions in two retinoschisis patients: We are providing clinical genetic testing to NEI clinics for X-lined Juvenile Retinoschisis. We sometime found patients with partial gene deletion. It was difficult to report back to patient each time without knowing the breakpoint of deletion and unable to carry out carrier analysis in mother and sisters. We had a couple of cases recently. We decided to analyze these cases for their genomic breakpoints. With help of a summer student, we have successfully determined the breakpoints and defined the deleted sequences. We are working on a manuscript to describe this study. To study OMD patients for mutations in gene RP1L1: Occult Macular Dystrophy is a very unique macular dystrophy. Clinical diagnosis is usually difficult and fully based on multifocal ERG. Recently, it was found that some Japanese patients with this disorder have mutations in a gene called RP1L1. Mutations in this gene have not found in Caucasians OMD patients and other RP patients at this moment. NEI ophthalmologists have been clinically evaluating patients with OMD. To explore NEI OMD patients for RP1L1 gene mutations, we have been developing a research procedure to screening the entire coding region of the RP1L1 gene. RP1L1 gene is a complicated gene that some parts of coding region are enriched with variable repetitive sequences, making it difficult in sequencing and data analysis. Dr. Xi Huang has been working on primer designing and procedure development last year. This summer, Ms. Julia Cao, a summer intern from Cornell University, has been working on sequencing of the RP1L1 genes in the NEI OMD patients. She has found the reported mutations in 3 of six index patients. We are continuing the analysis. We are planning to publish the study once we finish the mutation segregation analysis. To analyze promoter and intronic genomic abnormalities in Tyrosinase gene and collaboration with NHGRI Dr. Gahls group: This is also a continuation of my clinical research from Minnesota and collaboration with Dr. Bearing from NCI/NIH. I have been collecting tested samples for genetic heterogeneity analysis of TYR gene in the Ocular-Cutaneous Albinism (OCA). We had a research fellow who joined my lab early in 2010. She was working on this project since last year. She was focusing on the promoter region and had identified methylated CpG island through several approaches. She was working to correlate the methylation with regulation of gene expression at mRNA level through several cell lines and developing different approaches to identify enhancer/suppressor regions. She was working on sequencing of OCA2 gene. Last year we were also invited to join a multi-institute collaboration for the OCA study lead by Dr. David Adams of Dr. Gahls group from NHGRI. NEI DNA lab provides clinical tests of TYR gene and research sequencing analysis of OCA2 gene for the study. Unfortunately, our research fellow quitted from NEI because of personal visa issue recently. However, she is still working with us on summary of her results and we are planning to write a manuscript. To study GPR179 for CSNB: Very recently, a gene responsible for the Congenital Stationary Night Blindness (CSNB), GPR179, was identified. To study the GPR179 mutations for CSNB, a summer special volunteer, Ms. Ge Zhang, optimized a clinical protocol and sequenced 12 patients enrolled in NEI clinics. We did not find mutations in these patients.